This invention relates to methods for identifying substances capable of regulating the cell cycle. It further relates to the use of said substances in treating or preventing viral infection, cancer or cell death.
Herpes simplex virus (HSV) has a virulence determining locus in the long repeat region of its genome (Ackermann et al., 1986; Chou and Roizman, 1990; McGeoch et al., 1991; Dolan et al., 1992). The virulence phenotype has been specifically assigned to the RL1 gene and its encoded protein ICP34.5 (McKie et al., 1994). Null mutants in ICP34.5 are totally avirulent in mice (Taha et al., 1989a, b; Chou et al., 1990; MacLean et al., 1991) and the function of the protein in vitro has been shown to be cell type and cell state specific, depending on the stage in the cell cycle and the differentiation state (Brown et al., 1994).
One ICP34.5 function demonstrated in a human neuroblastoma cell line is the preclusion of host cell protein synthesis shut-off via the protein kinase PKR pathway following HSV infection (Chou and Roizman, 1992; Chou et al., 1995). This response to expression of ICP34.5 is however not ubiquitous and the precise molecular functions of ICP34.5 remain unknown.
A 63 amino acid carboxy terminal domain of ICP34.5 has been shown to share significant homology (McGeoch and Barnett, 1991) with the carboxy domain of the mouse myeloid differentiation protein MyD116 (Lord et al., 1990) and the hamster growth arrest and DNA damage gene GADD34 (Fornace et al., 1989) although the amino terminal parts of the proteins are quite diverse. The role of MyD116 and GADD34 in the cell appears to be in blocking growth and DNA replication following damage and thus they may act as tumour suppressor genes. The HSV type 1 (HSV1) strain 17 ICP34.5 protein comprises 248 amino acids whereas MyD116 and GADD34 are 657 and 590 amino acids respectively. Chou and Roizman (1994) have demonstrated that the carboxy terminus 63 amino acids are essential but not necessarily sufficient for the host cell shut-off phenotype of ICP34.5 and can be replaced by the homologous domain of MyD116 (Chou et al., 1996).
The present invention is based on the finding that ICP34.5 and MyD116 both interact, via their conserved domain, with proliferating cell nuclear antigen (PCNA). PCNA plays a role in several key cellular processes associated with cell cycle control and the maintenance of genome integrity. PCNA is involved in nucleotide excision repair where it associates with replication factor C and DNA polymerase xcex5 to form a component part of the DNA repair complexes. It is also involved in DNA replication where it acts as a processivity factor for eukaryotic DNA polymerase xcex4. Further, PCNA forms a complex with p21C1P1, an inhibitor of cyclin dependent kinases. The levels of p21 are up-regulated by the tumour suppressor p53, which is in turn activated by DNA damage and other forms of cellular stress.
Thus, the findings on which the present invention is based indicate that the role of HSV ICP34.5 may be to prevent host cell shut-down and/or cell death in response to cellular stress induced by viral infection, allowing viral replication to continue. In particular, an interaction between the C-terminus of ICP34.5 and PCNA may prevent or modify the interaction of PCNA with other components of the cell cycle control machinery which would normally result in host cell shut-down and/or cell death. One of these components may be MyD116/GADD34 and their homologues since ICP34.5 shares sequence homology with a region of MyD116/GADD34 and our results demonstrate that both ICP34.5 and MyD116 can bind PCNA.
Several possibilities arise from these findings. It may be possible to prevent viral propagation or the establishment of viral infection by disrupting the interaction between ICP34.5 and PCNA.
Thus the present invention provides a method for identifying a substance capable of disrupting an interaction between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) proliferating cell nuclear antigen (PCNA) or a homologue thereof, or a derivative thereof, which method comprises:
(a) providing an HSV ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, as a first component;
(b) providing PCNA or a homologue thereof, or a derivative thereof, as a second component;
(c) contacting the two components with a substance to be tested under conditions that would permit the two components to bind in the absence of the said substance; and
(d) determining whether the said substance disrupts the interaction between the first and second component.
The method of the invention may further comprise:
(e1) administering a said substance which has been determined to disrupt the interaction between the first and second components to a mammalian cell; and
(f1) determining the effect of the said substance on the cell cycle of the said cell.
The ability of the substance to induce cell cycle arrest may be determined. The ability of the substance to induce cell death by apoptosis may be determined.
Alternatively, the method of the invention may further comprise:
(e2) administering a virus to a cell in the absence of a said substance which has been determined to disrupt the interaction between the first and second components;
(f2) administering the virus to the cell in the presence of the said substance; and
(g2) determining if the said substance reduces or abolishes the susceptibility of the cell to viral infection.
The invention further provides a substance capable of disrupting an interaction between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) PCNA or a homologue thereof, or a derivative thereof, for use in treating the human or animal body by therapy or for use in diagnosis, whether or not practised on the human or animal body. Such a substance may thus be used in the prevention or treatment of viral infection. Preferably the target virus has homology to a herpes simplex virus. More preferably the target virus is a herpes simplex virus. Preferably the substance is identified by the method of the invention.
Since MyD116 and GADD34 have sequence homology with ICP34.5 and we have shown that MyD116 can bind to PCNA, it is likely that at least some of the activities of MyD116/GADD34 are mediated via similar interactions and pathways to ICP34.5. MyD116/GADD34 are thought to be involved in blocking cell growth and DNA replication following cellular stress, including DNA damage. Furthermore, we have shown that MyD116 is expressed in a range of different cell types of different species and that expression is not dependent on the differentiation state of the cell. Thus MyD116 is likely to have a conserved role in cell cycle regulation.
The invention therefore further provides a substance capable of disrupting between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) PCNA or homologues thereof, or derivatives thereof, for use in regulating the cell cycle of a mammalian cell. Again, preferably the substance is identified by the method of the invention. The substance may be used for inducing growth arrest and/or cell death. In that event, the mammalian cell is typically a tumour cell.
One function of ICP34.5 appears to be to prevent cell death induced by viral infection. This may be achieved by competing with MyD116/GADD34 or their homologues for PCNA. It may therefore be possible to prevent cell death in non-infected cells by inhibiting the activity of MyD116/GADD34 or their homologues. Thus the substance above may alternatively be used for preventing cell death. Preferably the cell is then a cell of the central or peripheral nervous system of a mammal, especially a human.
The invention also provides a method of regulating the cell cycle in a mammalian cell, which method comprises administering to said cell a substance capable of disrupting an interaction between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) PCNA or a homologue thereof, or a derivative thereof.
A further aspect of the invention relates to the identification of a novel human GADD34 homologue. The cellular GADD34 homologue is induced in response to HSV infection in permissive mammalian cells. Thus the invention provides a human GADD34 homologue which has one or more of the following features:
(i) a molecular mass of approximately 70 kDa as determined by SDS-PAGE;
(ii) a conserved region which is cross-reactive with an anti-ICP34.5 antibody;
(iii) cross-reactive with an anti-GADD34 antibody;
(iv) induced in permissive mammalian cells in response to HSV infection;
(v) not induced in permissive mammalian cells in response to heat shock or UV damage; and
(vi) not induced in non-permissive mammalian cells in response to HSV infection.
Preferably, the conserved region of the 70 kDa cellular homologue is at least 70% homologous with the C-terminal conserved region of hamster/human GADD34, more preferably at least 85% homologous. It is also preferred that the conserved region has a similar degree of homology with the C-terminal 63 amino acid residues of an HSV ICP34.5 polypeptide as that exhibited by MyD116/GADD34, preferably at least 30%. The conserved region over which the homology is compared is 30, preferably 50, more preferably 60 amino acids.
The human GADD34 homologue is only induced in response to HSV infection in permissive cells and not in non-permissive cells. Typically, induction of the human GADD34 homologue occurs at about 4 hours post-infection and plateaus at between 12 and 24 hours post-infection. Some HSV strains used therapeutically are attenuated to prevent establishment of a lytic replication cycle in non-permissive cell types and thus reduce their neurovirulence. For example, ICP34.5 negative strains are unable to replicate in fully differentiated, non-dividing neuronal cells. However, it may be desirable to use the lytic replication cycle of HSV in some therapeutic methods, for example cancer therapy. Thus it will be useful to determine easily whether a cell type is permissive or non-permissive for replication of attenuated HSV strains.
Our results indicate that the induction of the human GADD34 homologue may be used to determine whether a cell type will allow lytic replication by attenuated strains, for example, ICP34.5 negative HSV strains. In particular, it will be useful to determine if a tumour cell type is permissive or non-permissive. If the tumour cell-type is permissive, then it will be possible to administer ICP34.5 null mutants which will still replicate in the tumour cells. Lytic replication of attenuated HSV strains in the tumour cells may improve the tumour killing properties of the HSV strains.
Thus the invention provides a method for determining whether a cell is permissive for HSV lytic replication which method comprises:
(a) infecting said cell with wild-type HSV and
(b) determining if the 70 kDa cellular GADD34 homologue is induced.
Typically, induction of the GADD34 homologue is determined by Western blotting cellular extracts. Preferably the cell is a human tumour cell.
POLYPEPTIDE COMPONENTS
The first component comprises a ICP34.5 polypeptide or a homologue thereof or a derivative of ICP34.5 or of an ICP34.5 homologue. Homologues of ICP34.5 include MyD116 and GADD34. Derivatives of ICP34.5 include fragments of ICP34.5, MyD116 and GADD34 which comprise at least a region having substantial homology to the C-terminal 63 amino acids of ICP34.5. The fragments may be up to 63, 70, 80, 90 or 100 amino acid residues long. The minimum fragment length may be 6, 10, 20 or 30 amino acid residues. Herein, substantial homology for fragments of ICP34.5 is regarded as a sequence which has at least 70%, e.g. 80%, 90% or 95%, amino acid homology (identity) over 30, preferably 50, more preferably 60 amino acids with the C-terminal 63 amino acids of ICP34.5. Substantial homology for fragments of MyD116 and GADD34 is regarded as a sequence which has a similar degree of homology with the C-terminal 63 amino acid residues of an HSV ICP34.5 polypeptide as that exhibited by MyD116/GADD34, preferably at least 30%. Derivatives further include variants of ICP34.5 and its homologues or derivatives, including naturally occurring allelic variants and synthetic variants which are substantially homologous to said ICP34.5 and its homologues. The sequence of HSV ICP34.5 is described in Chou and Roizman, 1990; Dolan et al., 1992 and McGeoch et al., 1991.
Derivatives of ICP34.5 and its homologues may contain one or more (e.g. 2, 3, 5 or 10) substitutions, deletions or insertions, including conserved substitutions. Conserved substitutions may be made according to the following table which indicates conservative substitutions, where amino acids on the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Derivatives may be in the form of a fusion protein wherein ICP34.5, a homologue or derivative thereof is fused, using standard cloning techniques, to another polypeptide which may, for example, comprise a DNA-binding domain, a transcriptional activation domain or a ligand suitable for affinity purification (for example glutathione-S-transferase or six consecutive histidine residues).
The second component is selected from PCNA or homologues thereof, and their derivatives. Preferably the PCNA is mammalian PCNA, more preferably human PCNA. Derivatives of PCNA include fragments, preferably comprising at least 30 amino acids, more preferably at least 50 amino acids, which are capable of binding to ICP34.5. Derivatives further include variants of PCNA, its homologues or derivatives, including naturally occurring allelic variants and synthetic variants which are substantially homologous to said PCNA. In this context, substantial homology is regarded as a sequence which has at least 70%, e.g. 80% or 90% amino acid homology (identity) over 30, preferably 50, more preferably 60 amino acids with PCNA.
Derivatives of PCNA and its homologues may contain one or more (e.g. 2, 3, 5 or 10) substitutions, deletions or insertions, including conserved substitutions. Conserved substitutions may be made according to the table represented and described above. Derivatives may be in the form of a fusion protein wherein said PCNA, homologue or derivative thereof is fused to another polypeptide which may, for example, comprise a DNA-binding domain, a transcriptional activation domain or a ligand suitable for affinity purification (for example glutathione-S-transferase or six consecutive histidine residues).
The first and second components used in the assays may be obtained from mammalian or yeast cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines. Preferably, the first and second components used in the assays are recombinant.
CANDIDATE SUBSTANCES
A substance which disrupts an interaction between the first component (a polypeptide selected from an HSV ICP34.5 polypeptide or a homologue thereof, or a derivative thereof) and the second component (PCNA or its homologues, and derivatives thereof) may do so in several ways. It may directly disrupt the binding of the two components by, for example, binding to one component and masking or altering the site of interaction with the other component. Candidate substances of this type may conveniently be screened by in vitro binding assays as, for example, described below. Examples of candidate substances include non-functional homologues of the first or second components as well as antibodies which recognise the first or second components.
A substance which can bind directly to the first or second component may also inhibit an interaction between the first component and the second component by altering their subcellular localisation thus preventing the two components from coming into contact within the cell. This can be tested in vivo using, for example the in vivo assays described below. The term xe2x80x98in vivoxe2x80x99 is intended to encompass experiments with cells in culture as well as experiments with intact multicellular organisms.
Alternatively, instead of preventing the association of the components directly, the substance may suppress or enhance the biologically available amount of one or both of the components. This may be by inhibiting expression of the component, for example at the level of transcription, transcript stability, translation or post-translational stability. An example of such a substance would be antisense RNA which suppresses the amount of MyD116 mRNA translated into protein.
Suitable candidate substances include peptides, especially of from about 5 to 20 amino acids in size, based on the sequence of the conserved C-terminal domain of ICP34.5/MyD116/GADD34, or variants of such peptides in which one or more residues have been substituted. Peptides from panels of peptides comprising random sequences or sequences which have been varied consistently to provide a maximally diverse panel of peptides may be used.
Suitable candidate substances also include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) which are specific for the first component or the second component, preferably the conserved C-terminal domain of ICP34.5/MyD116/GADD34. Furthermore, combinatorial libraries, peptide and peptide mimetics, defined chemical entities, oligonucleotides, and natural product libraries may be screened for activity as inhibitors of an interaction between the first component and the second component in assays such as those described below. The candidate substances may be used in an initial screen in batches of, for example 10 substances per reaction, and the substances of those batches which show inhibition tested individually. Candidate substances which show activity in in vitro screens such as those described below can then be tested in in vivo systems, such as mammalian cells which will be exposed to the inhibitor and tested for susceptibility to viral infection or apoptosis as appropriate.
Assays
The assays of the invention may be in vitro assays or in vivo assays, for example using an animal model. One type of in vitro assay for identifying substances which disrupt an interaction between the first component and the second component involves:
contacting a first component, which is immobilised on a solid support, with a non-immobilised second component in the absence of a candidate substance;
contacting the first immobilised component with the non-immobilised second component in the presence of a candidate substance; and
determining if the candidate substance disrupts the interaction between the first component and the second component.
Alternatively, the second component may be immobilised and first component non-immobilised.
In a preferred assay method, the first component is immobilised on beads such as agarose beads. Typically this is achieved by expressing the component as a GST-fusion protein in bacteria, yeast or higher eukaryotic cell lines and purifying the GST-fusion protein from crude cell extracts using glutathione-agarose beads (Smith and Johnson, 1988). As a control, binding of the second component, which is not a GST-fusion protein, to the immobilised first component is determined in the absence of the candidate substance. The binding of the second component to the immobilised first component is then determined in the presence of the candidate substance. Any inhibitory effect by the candidate substance can then be evaluated. This type of assay is known in the art as a GST pulldown assay.
The candidate substance may be pre-incubated with the first component or with the second component or added to the reaction mixture after pre-incubation of the first component with the second component. In a similar assay, the second component is a GST fusion protein immobilised on glutathione agarose beads and the first component is a not a GST-fusion protein. It is also possible to perform this type of assay using different affinity purification systems for immobilising one of the components, for example Ni-NTA agarose and histidine-tagged components.
Binding of the first component to the second component (and vice-versa) may be determined by a variety of methods well-known in the art. For example, the non-immobilised component may be labelled (with for example, a radioactive label, an epitope tag or an enzyme-antibody conjugate). The effect of a candidate substance on an interaction between the two components can be determined by comparing the amount of label bound in the presence of the candidate substance with the amount of label bound in the absence of candidate substance. A lower amount of label bound in the presence of the candidate substance indicates that the candidate substance is an inhibitor of interactions between the first component and the second component.
Alternatively, binding may be determined by immunological detection techniques. For example, the reaction mixture can be Western blotted and the blot probed with an antibody that detects the non-immobilised component. ELISA techniques may also be used.
Another method contemplated by the invention for identifying a substance that disrupt an interaction between the first component and the second component involves immobilising the first component on a solid support coated (or impregnated with) a fluorescent agent, labelling the second component with a substance capable of exciting the fluorescent agent, contacting the immobilised first component with the labelled second component in the presence and absence of a test compound, detecting light emission by the fluorescent agent, and identifying inhibitory substances as those candidate substances that reduce the emission of light by the fluorescent agent in comparison to the emission of light by the fluorescent agent in the absence of the test compound. Alternatively, the second component may be immobilised and first component labelled in the assay.
Assays for identifying compounds that disrupt an interaction between the first and second component may involve:
(a) transforming or transfecting an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain;
(b) expressing in the host cell a first hybrid DNA sequence encoding a first fusion of all or part of the first component and the DNA binding domain or the activating domain of the transcription factor; expressing in the host cells a second hybrid DNA sequence encoding all or part of the second component and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion;
(c) evaluating the effect of a test compound on the interaction between the first component and the second component by detecting binding of the first component to the second component in a particular host cell by measuring the production of reporter gene product in the host cell in the presence or absence of the test compound; and
(d) determining whether the presence of the test compound alters the production of the reporter gene product in comparison to the production of the reporter gene product in the absence of the test compound.
The host cell may be a bacterium or other microbial cell. It may be a yeast or mammalian cell. Presently preferred for use in such an assay are a lexA promoter to drive expression of the reporter gene, the lacZ reporter gene, a transcription factor comprising the lexA DNA domain and the GAL4 transactivation domain and yeast host cells.
The candidate substance, i.e. the test compound, may be administered to the cell in several ways. For example, it may be added directly to the cell culture medium or injected into the cell. Alternatively, in the case of polypeptide candidate substances, the cell may be transfected with a nucleic acid construct which directs expression of the polypeptide in the cell. Preferably, the expression of the polypeptide is under the control of a regulatable promoter.
Candidate substances that are identifiable by the method of the invention as disrupting an interaction between a first component and a second component may be tested for their ability to, for example, reduce susceptibility of cells to viral infection or regulate the cell cycle including apoptosis and growth arrest. Such compounds could be used therapeutically to prevent or treat viral infection. They may also be used therapeutically in regulating the cell cycle of a mammalian cell, including preventing cell death in, for example, neuronal cells, or inducing cell death in, for example, neoplastic cells.
Typically, an assay to determine the effect of a candidate substance identifiable by the method of the invention on the susceptibility of cells to viral infection comprises:
(a) administering a virus, for example HSV1, to a cell, for example a BHK21/C13 cell, in the absence of the candidate substance;
(b) administering the virus to the cell in the presence of the candidate substance; and
(c) determining if the candidate substance reduces or abolishes the susceptibility of the cell to viral infection.
The candidate substance may be administered before, or concomitant with, the virus to establish if infection is prevented. Alternatively, the candidate substance may be administered subsequent to viral infection to establish if viral infection can be treated using the candidate substance. Administration of candidate substances to cells may be performed as described above.
The assay is typically carried out in vitro but an animal model could be employed instead. The virus is contacted with cells, typically cells in culture. The cells may be cells of a mammalian cell line, in particular mammalian cells susceptible to infection by the virus in the absence of the candidate substance.
Techniques for assaying infectivity of viruses are well-known in the art. As well as using plaque assays, levels of viral infection can be determined by using recombinant viruses which comprise a reporter gene, for example lacZ. The use of a histochemically detectable reporter gene is especially preferred when experiments are performed with animals, for example mice.
Typically, an assay to determine the effect of a candidate substance identifiable by the method of the invention on the regulation of the cell cycle in a mammalian cell comprises:
(a) administering the candidate substance to the cell; and
(b) determining the effect of the candidate substance on the cell cycle, including, for example induction of cell cycle arrest and/or cell death by apoptosis.
Administration of candidate substances to cells may be performed as described above. The assay is typically carried out in vitro. The candidate substance is contacted with the cells, typically cells in culture. The cells may be cells of a mammalian cell line.
The ability of a candidate substance to induce apoptosis can be determined by administering a candidate compound to cells and determining if apoptosis is induced in said cells. The induction of apoptosis can be determined by various means. There are several techniques known to a skilled person for determining if cell death is due to apoptosis. Apoptotic cell death is characterised by morphological changes which can be observed by microscopy, for example cytoplasmic blebbing, cell shrinkage, internucleosomal fragmentation and chromatin condensation. DNA cleavage typical of the apoptotic process can be demonstrated using TUNEL and DNA ladder assays.
Alternatively, it may be desired to prevent apoptotic cell death by administering a substance identifiable by the method of the invention which prevents an interaction between MyD116 or GADD34 and their homologues, and PCNA. Several techniques known in the art for inducing apoptosis in cells may be used. For example, apoptosis may be induced by stress including UV exposure, growth factor deprivation and heat shock. The ability of the candidate substance to inhibit such apoptosis may be determined by comparing cells exposed to stress in the presence of the candidate substance with those exposed to stress in the absence of the candidate substance.
In a preferred embodiment of the above-described assays, ICP34.5 and derivatives thereof are used in an experimental system to study normal cellular interactions. For example, derivatives of ICP34.5, including deletion, insertion and substitution mutants, can be used to disrupt an interaction between MyD116 and PCNA. This can be tested in vitro using the in vitro assays described above. The interaction between MyD116 and PCNA can also be disrupted in vivo by introducing ICP34.5 and derivatives thereof, including deletion, insertion and substitution mutants, into cells in vivo, preferably mammalian cells, more preferably human cells. ICP34.5 and its derivatives can be introduced into the cells using techniques described above, for example transfection of nucleic acid constructs encoding ICP34.5 and its derivatives, or using viral vectors, preferably HSV. The effect of this disruption can be determined using immunoprecipitation studies or, alternatively, by analysing the effect on cell cycle control using, for example, the assays and techniques described above. Any in vitro data obtained may be used to assist in the rational design of ICP34.5 derivatives for use in the in vivo studies. In addition, the precise regions/amino acid residues of ICP34.5 which bind to PCNA can determined by in vitro binding studies using ICP34.5 derivatives and PCNA. This will also assist in the rational design of ICP34.5 derivatives for use in the in vivo studies.
Thus ICP34.5 and its derivatives, which are readily distinguished from cellular constituents, may be used as a tool to investigate cell cycle control.
The induction of the 70 kDa cellular homologue of GADD34 in cells by infection with wild-type HSV may be used to determine if a cell is permissive for HSV lytic replication.
A typical assay comprises:
(a) infecting said cell with wild-type HSV and
(b) determining if the 70 kDa cellular GADD34 homologue is induced.
Induction of the 70 kDa cellular GADD34 homologue may be determined by, for example, Western blotting cellular extracts. For example, extracts from uninfected cells and cells infected with wild-type HSV are resolved by SDS-PAGE, immunoblotted and probed with an anti-GADD34 antibody. If the homologue has been induced, an approximately 70 kDa cross-reactive band should be present in the infected cell extracts but not the uninfected cell extracts. It is also possible to use extracts of cells infected with an ICP34.5 null HSV mutant as the negative control.
The cells are preferably human tumour cells and are typically obtained from tissue biopsies of patients"" tumours.
Therapeutic Uses
An essential part of the HSV infection process appears to be preventing host cell shutdown in response to the infection. We have shown that the mechanism for this may be an interaction between ICP34.5 and components of the cell cycle regulatory apparatusxe2x80x94PCNA. Thus the present invention provides a substance capable of disrupting an interaction between (i) an HSV ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) a polypeptide selected from proliferating cell nuclear antigen or homologues thereof, or derivatives thereof, for use in a method of preventing or treating viral infection.
Further, since HSV ICP34.5 and MyD116 appear to be involved in cell cycle regulation through their interaction with PCNA, such a substance may be used to regulate the cell cycle of a mammalian cell. Thus the present invention provides a substance capable of disrupting an interaction between (i) an HSV ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) PCNA selected or homologues thereof, or derivatives thereof, for use in a method of regulating the mammalian cell cycle. Typically, said substance may be used to induce cell death, for example in a tumour cell, or to prevent cell death, in for example a cell of the central or peripheral nervous system.
The formulation of a substance according to the invention will depend upon the nature of the substance identified but typically a substance may be formulated for clinical use with a pharmaceutically acceptable carrier or diluent. For example it may formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration. A physician will be able to determine the required route of administration for any particular patient and condition.
Preferably, the substance is used in an injectable form. It may therefore be mixed with any vehicle which is pharmaceutically acceptable for an injectable formulation, preferably for a direct injection at the site to be treated. The pharmaceutically carrier or diluent may be, for example, sterile or isotonic solutions. It is also preferred to formulate that substance in an orally active form. Typically, said substance may be a polypeptide, an antibody or a nucleic acid construct. Nucleic acid constructs may be administered by various well-known techniques including lipofection, biolistic transformation or the use of viral vectors.
The dose of substance used may be adjusted according to various parameters, especially according to the substance used, the age, weight and condition of the patient to be treated, the mode of administration used and the required clinical regimen. A physician will be able to determine the required route of administration and dosage for any particular patient and condition.
The invention will be described with reference to the following Example which is intended to be illustrative only and not limiting. In the accompanying drawings: